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Alcoholism and the Brain Reward Pathway

Alcoholism and the Brain Reward Pathway. Mentor: Dr. Michael Miles PhD Candidate: Jennifer Wolstenholme-Faison Shauna Spanos July 23, 2007. Goals. To Drink or not to Drink? Determine molecular and behavioral variations My project

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Alcoholism and the Brain Reward Pathway

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  1. Alcoholism and the Brain Reward Pathway Mentor: Dr. Michael Miles PhD Candidate: Jennifer Wolstenholme-Faison Shauna Spanos July 23, 2007

  2. Goals • To Drink or not to Drink? • Determine molecular and behavioral variations • My project • Identify areas of the brain that are activated/excited upon alcohol consumption • Looking for verification • Ultimately: • Develop drug to prevent relapse in alcoholics

  3. Alcoholism • Seeking alcohol despite negative effects • In the work place • Socially and personally • Excessive intake of alcohol • Altered Brain Reward Pathway • Allostasis versus Homeostasis http://aboutalcoholism.biz/drunk.jpg

  4. Brain Reward Pathway • Ventral Tegmental Area (VTA) releases dopamine • Nucleus Accumbens contains dopamine sensitive cells • Causes feelings of pleasure • Amygdala and Hippocampus (not shown) plays role in memory and whether experience is desirable • Prefrontal Cortex coordinates all the information and determines behavior of individual • Pathway the same for opiates, methamphetamines, and nicotine

  5. What We’re Looking For • Transcription Factors • Proteins that up-regulate or down-regulate the expression of other genes • Specifically interested in the c-Fos TF • c-FOS is an Immediate Early Gene (IEG) • Expressed rapidly after cellular stimuli • Looking for region of brain where c-FOS most abundant • Our Expectation • To find differences in cFOS expression between high drinking mice and low drinking mice

  6. My Project • 20 C57BL/6NCrl Mice from Charles Rivers Laboratory • because they like to drink • 2 choices – 10% ethanol or water • Monitored drinking behavior for 2 weeks • Unlimited access to alcohol • Model without influences of stress • Highly Inbred • variation in drinking behavior http://www.uwo.ca/biology/Faculty/singh/images/drinkingmouse.jpg

  7. The Sacrifice • Day 14 • Recorded Data • Sacrificed Mice • Gassed mice with CO2 • Fixed brains in 4% Paraformaldehyde in phosphate-buffered saline (PBS)

  8. The Data Don’t Peek

  9. Slices for Slides • Cryostat slices brain • Temp ~ -10 to -20 C • 40 μm slices • Stored in PBS (Phosphate Based Saline) • Dispose Olfactory Bulbs

  10. Staining • Stain every 6th slice • Single cell is approximately 20 μm deep • 40 μm slices, every 6th slice • Viewing new cell in each slice

  11. Analysis - Immunohistochemistry • Goat Serum for blocking • Rabbit polyclonal primary antibody attaches to N-terminus of cFOS protein • Goat anti-rabbit antibody attaches • Avidin-biotin-horseradish peroxidase complex attaches • Result is brown staining visible on the slice NH2-------COOH

  12. Results

  13. Results 20x, Lateral Septum

  14. Results 20x, Hippocampus

  15. What Now. . . • Quantify Data • Is there a difference in cFOS expression between high drinkers and low drinkers? • If there is a difference • Did it cause the drinking variation? • Or, was it a result of the drinking variation? • Redo Experiment • Change variables

  16. Acknowledgements • Thank you Dr. Miles and Jennifer • Thank you Nate, Sean, Aaron, Alex and Ryan

  17. References • Wightman, Mark. New insights on the neural basis of brain reward and alcohol drinking. UNC Bowles Center for Alcohol Studies Center Line, Volume 11, Number 2, 2000. Retrieved June 12, 2007 from http://www.med.unc.edu/alcohol/cenline/11_2_1.htm. • Koob, George F. (2003). Alcoholism: Allostasis and Beyond. Alcoholism: Clinical and Experimental Research, Vol.27, No. 2, February 2003. • Hill, Katherine G., et al. FOS expression induced by an ethanol-paired conditioned stimulus. Pharmacology Biochemistry and Behavior (2007), doi:10.1016/j. Pbb.2007.04.017 • Jackson Laboratory. http://jaxmice.jax.org/info/index.html

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